Mycosphere 8(7): 827–834 (2017) www.mycosphere.org ISSN 2077 7019 Article Doi 10.5943/mycosphere/8/7/2 Copyright © Guizhou Academy of Agricultural Sciences Sarocladium brachiariae sp. nov., an endophytic fungus isolated from Brachiaria brizantha Liu XB1, Guo ZK2 and Huang GX1* 1Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS), Key Laboratory of Integrated Pest Management on Tropical Crops, Ministry of Agriculture, Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Haikou, Hainan 571101, P. R. China. 2 Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (CATAS), Haikou, Hainan 571101, P. R. China. Liu XB, Guo ZK, Huang GX 2017 – Sarocladium brachiariae sp. nov., an endophytic fungus isolated from Brachiaria brizantha. Mycosphere 8(7), 827–834, Doi 10.5943/mycosphere/8/7/2 Abstract In a survey on the diversity of endophytic fungi of Brachiaria brizantha, a new species of Sarocladium was isolated and proposed here as Sarocladium brachiariae. According to the LSU and ITS rDNA sequences and culture morphology and micromorphology, the species differed from the species hitherto described in Sarocladium, and is characterized as a new species. The new species can produce hyphal coils and slimy heads. In addition, raised, cottony, moist to slimy colonies on PDA, and adelophialides, branched conidiophores were also useful characters for distinguishing species from other species of Sarocladium. Keywords – Endophytes – Sarocladium – Phylogeny – Taxonomy Introduction Sarocladium was established by Gams and Hawksworth (1976) based on a species described by Sawada in Taiwan, firstly including two fungal pathogens causing sheath rot of rice. Based on a recent molecular phylogenetic study of rDNA sequences, Summerbell et al. (2011) transferred seven species, formerly named Acremonium kiliense, A. strictum, A. zeae, A. bacillisporum, A. bactrocephalum, A. glaucum and A. ochraceum, to the genus. Yeh & Kirschner (2014) introduced a new species named Sarocladium spinificis. Giraldo et al. (2015) used multilocus phylogenetic inferences combined with phenotypic data, to introduce S. bifurcatum, S. gamsii, S. hominis, S. pseudostrictum, S. subulatum and S. summerbellii, and re-allocated A. implicatum and A. terricola to the genus. Sarocladium attenuatum is confirmed as synonym of the type species of the genus, S. oryzae. Sarocladium presently encompasses 18 species (Summerbell et al. 2011, Giraldo et al. 2015). Although Sarocladium species have traditionally been considered as important phytopathogens (Gams and Hawksworth 1976, Ayyadurai et al. 2005), the genus also contains opportunistic human pathogens (Fincher et al. 1991, Perdomo et al. 2011), and other properties of applied value. For example, S. oryzae produces antibiotics (Bills et al. 2004), and S. zeae (Gams & Sumner, 1971) is considered as a mutualistic endophyte in maize (Wicklow et al. 2008). Endophytic fungi form nonpathogenic and intercellular associations with host, completing their entire life cycle in the plant Submitted 29 March 2017, Accepted 19 May 2017, Published 9 June 2017 Corresponding Author: Gui-Xiu Huang – e-mail – [email protected] 827 (Kelemu et al. 2001). Species of the genus Sarocladium are commonly associated with members of Poaceae (Yeh & Kirschner 2014, Giraldo et al. 2015). Some endophytic fungi are potential biological control agents for plant pathogens (Kelemu et al. 2001). Endophytic-infected plants also possess other properties such as growth stimulation, improved survival, and drought tolerance (Lim et al. 2000, Yang et al. 2014). Brachiaria are commercially important forage grasses in tropical area. In this study, we collected endophytic fungi associated with Brachiaria spp. in China, and a species was identified as a member of Sarocladium, but was not identical to any of the previously described species. Materials & Methods Plant tissue staining, fungal isolations, and culture maintenance In 2005, healthy grass leaves and sheaths of Brachiaria brizantha were collected from Danzhou, China. Plant tissue staining and endophytic fungal isolation were conducted according to the method described by Kelemu et al. (2001). Samples which appeared to have intercellularly growing hyphae were noted, and plants from which the samples originated were used for further fungal isolation. Small pieces (5 mm) of tissues that surface sterilized were plated on potato- dextrose agar, and incubated for 4 to 6 weeks at 28 ºC. Cultures were effectively maintained either on PDA supplemented with 10 µg/mL tetracycline, or, for long-term storage, by lyophilization according to the method described by Kelemu et al. (2001). DNA extraction, amplification and sequencing Isolates grow on PDA for 10 days at 25 ºC. Genomic DNA was extracted using Genomic DNA Spin Kit (Plant), according to the modified manufacturer’s protocol (TIANGEN Co., Ltd., China). The internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) and D1/D2 domains of the large-subunit nrRNA were amplified with the primer pairs ITS1/ITS4 and NL1/NL4b, respectively (White et al. 1990, O’Donnell 1993). The PCR products were visualised on an electrophoresis gel after GoldView (Solarbio, China) staining. PCR products of the expected size were purified with E.Z.N.A.TM Gel Extraction Kit I (OMEGA) according to the manufacturer’s instructions. DNA sequencing was done by BGI Biotech (Shenzhen, China) with 3730 DNA Analyzer (Applied Biosystems). Alignment and phylogenetic analysis DNA Sequences were assembled and edited using SeqMan II software (DNAStar, Inc., Madison, Wis.). Related DNA sequences of ITS and LSU rDNA were compared using the BLAST function of GenBank. For phylogenetic analysis, sequences retrieved from the BLAST search and the taxon sampling based on ITS and LSU rDNA sequences (Yeh & Kirschner 2014, Giraldo et al. 2015) were used (Table1). In addition to sequences of Sarocladium spp., Acremonium species closely related with Sarocladium spp., but outside the Sarocladium clade, were included as outgroup (Giraldo et al. 2012). Multiple sequence alignments were performed with Clustal W using MEGA 5 (Tamura et al. 2011) and manually corrected where necessary. Nucleotide substitution models were generated using MrModeltest v. 2.3 (Nylander et al. 2008). A maximum likelihood phylogenetic analysis of the dataset was performed with PAUP v.4.0b10 (Swofford 2002). Markov Chain Monte Carlo (MCMC) sampling was used to reconstruct phylogenies in Mrbayes v. 3.2 (Ronquist & Huelsenbeck 2003). Analyses of 2 MCMC chains based on the full dataset were run for 1 × 107 generations and sampled every 100 generations. The alignments and trees in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S20969?x-access- code=e79cef78c47c3d70ebcb 7314c8a991ab&format=html), and taxonomic novelties in MycoBank (Crous et al. 2004). 828 Phenotypic studies Morphological characterization of the fungal isolates was carried out based on cultures grown on potato dextrose agar (PDA), oatmeal agar (OA; filtered oat flakes after 1 h of simmering 30 g, agar 20 g, distilled water to final volume of 1000 mL) and malt extract agar (malt extract 20 g, peptone 1 g, glucose 20 g, agar 15 g, H2O 1000 ml). Cultures were incubated at 25 ± 1 ºC in the dark and periodically examined each 7 days up to 4 weeks. Colony diameters were measured after 14 days of growth, and colony colours determined using the colour charts of Kornerup & Wanscher (1978). In addition, the ability of the isolates to grow at 15, 20, 25, 30, 35, 37 and 40 ºC was determined on PDA. Conidiophore morphology was observed on cultures grown on dilute malt agar (malt extract 5 g, 12 g agar, 1000 ml distilled H2O) supported on microscope coverslips, other microscopic features were examined and measured by slide cultures on OA, using an Ni-E light microscope (Nikon Corporation, Japan). Photomicrographs were made with a Nikon DS-Ri1 light microscope (Nikon Corporation, Japan). Scanning electron microscope (SEM) micrographs were obtained with a S-3000N scanning electron microscope (Hitachi, Japan). Results Phylogenetic analysis The BLAST query revealed that the isolate was shown to be closely related to Sarocladium on the basis of its D1/D2 and the ITS regions. Sequences of the D1/D2 (889 bp) and ITS (585 bp) were deposited in GenBank as KP715271 and EU880834, respectively. When comparing the BLAST search results among sequences exceeding a length of 831 bp of D1/D2 fragments, the highest similarity between our isolate and other identified Sarocladium species was 99%, namely with one strain of Sarocladium oryzae (GenBank number HG965047, Giraldo et al. 2015). The highest similarity of ITS rDNA fragments was 95%, namely with one strain of Sarocladium kiliense (Genbank number LN864540, Asadzadeh 2015). The systematic positions of Sarocladium sp. was estimated by the combined analysis from ITS and D1/D2 partial gene consisted of 980 characters including alignment gaps. The phylogenetic analysis allowed distributing the isolates included in this study into 18 lineages (Fig. 1). These lineages were phylogenetically distant enough to be considered as different species. Taxonomy On the basis of phylogenetic analysis and phenotypic features, we propose that the isolated fungus Sarocladium sp. is different from any previously described species in this genus and therefore are proposed as new. Sarocladium brachiariae X.B. Liu, G.X. Huang & Z.K Guo sp. nov. Fig. 2A-K MycoBank